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This article includes Supplementary Figures available at http://www.interscience.wiley.com/jpages/0270-9139/suppmat

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jwsHEPv42.1.200.fig1.pdf86K Phosphorylation, protein levels and effect of inhibition of EGF receptor during H2O2 exposure. <br /> <br /> (A) Primary cultures of hepatocytes were stimulated with increasing amounts of H2O2 and EGF for 10 minutes, and subjected to Western immunoblotting analysis utilizing antibodies that recognize EGF receptor phosphorylated at Y1173.<br /> <br /> (B) Hepatocytes were incubated with increasing amounts of the EGF receptor inhibitor PD153035 for 2 hours, and stimulated with 1 mM H2O2 or 10 nM EGF for 10 minutes. The cells were harvested and subjected to Western immunoblotting utilizing antibodies against phosphorylated ERK.<br /> <br /> (C) Hepatocytes were stimulated with 1 mM H2O2 or 10 nM EGF for 10 minutes. The cells were harvested and subjected to Western immunoblotting utilizing antibodies that recognizes EGF receptor phosphorylated at Y845.<br /> <br /> (D) Hepatocytes were stimulated with 1 mM H2O2 or 10 nM EGF for various time points, as shown, and subjected to Western immunoblotting analysis utilizing antibodies against the EGF receptor. In A-D, the protein expression of β-tubulin was used as a loading control. Representative examples of at least three experiments are shown.
jwsHEPv42.1.200.fig2.pdf447K Immunostaining of the EGF receptor after stimulation with H2O2 and EGF. <br /> <br /> Hepatocytes were stained with anti-EGF receptor antibodies (green color) combined with anti-EEA1antibodies (red color) in control cells (A), and in hepatocytes 10 minutes after stimulation with EGF (B) and H2O2 (C). Merged pictures are shown and colocalization between red and green color yield yellow color. Representative images of at least three experiments are shown.
jwsHEPv42.1.200.fig3.pdf540K Immunofluorescence staining of MEK after treatment of cell with Leptomycin B. <br /> <br /> Hepatocytes were treated with EGF or H2O2 prior to immunostaining with anti-MEK. Confocal laser scan images of MEK are shown. Hepatocytes with (B, D, F) or without (A, C, E) 30 minutes preincubation with 2 ng/ml Leptomycin B. (A, B) control hepatocytes (C, D) 10 nM EGF for 10 minutes (E, F) 1 mM H2O2 for 10 minutes. Representative images of at least three experiments are shown.
jwsHEPv42.1.200.fig4.pdf66K Time-dependent phosphorylation of ERK during H2O2 and EGF-exposure. <br /> <br /> Primary cultures of hepatocytes were stimulated with 1 mM H2O2 and 10 nM EGF for the time points indicated. The cells were harvested, and Western immunoblotting analysis was performed on hepatocyte lysates with an antibody that recognizes phosphorylated ERK, or with an anti-ERK1 antibody recognizing ERK1 and ERK2. The protein expression of β-tubulin was used as a loading control. A representative example of at least three experiments is shown.
jwsHEPv42.1.200.fig5.doc32K DNA synthesis in hepatocytes stimulated with H2O2 or EGF. <br /> <br /> [3H]thymidine incorporation was measured at 72 hours in hepatocytes stimulated with increasing amounts of H2O2 (0.1 mM; 0.5 mM; 1 mM; 5 mM) or EGF (0.01 nM; 0.1 nM; 1nM; 10 nM). SEM, n=3

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